Legume Genomics and Genetics 2025, Vol.16 http://cropscipublisher.com/index.php/lgg © 2025 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved.
Legume Genomics and Genetics 2025, Vol.16 http://cropscipublisher.com/index.php/lgg © 2025 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. CropSci Publisher is an international Open Access publishing specializing in crop genome, trait-controlling, crop gene expression and regulation at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. . Publisher Cropsci Publisher Edited by Editorial Team of Legume Genomics and Genetics Email: edit@lgg.cropscipublisher.com Website: http://cropscipublisher.com/index.php/lgg Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Legume Genomics and Genetics (ISSN 1925-1580) is an open access, peer reviewed journal published online by CropSci Publisher. The journal is committed to publishing grain/forage legume studies, as well as research on model legume plants such as Lotus japonicus and Medicago truncatula. The aims are to feature innovative research findings in the basic and applied fields of legume biology. Topics include (but are not limited to) genome structure, genome-scale analysis, comparative and functional genomics, proteomics and epigenomics, gene discovery and function, gene expression and evolution, as well as legume genetics from the molecular level to whole plant level. All the articles published in Legume Genomics and Genetics are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CropSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Legume Genomics and Genetics (online), 2025, Vol. 16, No.5 ISSN 1925-1580 http://cropscipublisher.com/index.php/lgg © 2025 CropSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Expansin Gene Family in Legumes: Structural Diversity and Expression Dynamics Dandan Huang Legume Genomics and Genetics, 2025, Vol.16, No.5, 204-214 Gene Editing-Assisted Development of Herbicide-Resistant Lentils Hongpeng Wang, Shiying Yu Legume Genomics and Genetics, 2025, Vol.16, No.5, 215-224 Role of Epigenetic Modifications in Regulating Nodule Development Jin Wang, Lin Liu, Congbiao You Legume Genomics and Genetics, 2025, Vol.16, No.5, 225-233 Molecular Dissection of Cold Response Pathways in Adzuki Bean Xiaoxi Zhou, Tianxia Guo Legume Genomics and Genetics, 2025, Vol.16, No.5, 234-244 Comparative Analysis of Anti-Nutritional Factors in Edible Legumes Weiliang Shen, Dan Luo, Xinhua Zhou Legume Genomics and Genetics, 2025, Vol.16, No.5, 245-252
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 204 Research Insight Open Access Expansin Gene Family in Legumes: Structural Diversity and Expression Dynamics Dandan Huang Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: dandan.huang@hibio.org Legume Genomics and Genetics, 2025 Vol.16, No.5 doi: 10.5376/lgg.2025.16.0021 Received: 03 Jul., 2025 Accepted: 20 Aug., 2025 Published: 05 Sep., 2025 Copyright © 2025 Huang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Huang D.D., 2025, Expansin gene family in legumes: structural diversity and expression dynamics, Legume Genomics and Genetics, 16(5): 204-214 (doi: 10.5376/lgg.2025.16.0021) Abstract Expansins are plant-specific proteins that play crucial roles in cell wall loosening and are essential for various developmental processes and stress responses. This study comprehensively analyzes the expansin gene family in legumes, focusing on its structural diversity, evolutionary patterns, expression dynamics, and functional relevance. We classify expansins into α-expansin, β-expansin, and expansin-like A and B subfamilies and discuss their gene duplication events, chromosomal localization, and conserved domain structures in major legume species. Phylogenetic relationships and Ka/Ks ratio analyses provide new insights into their evolutionary trajectories and selective pressures. Furthermore, we investigate tissue-specific and developmental stage-specific expression patterns, highlighting the roles of expansins in root growth, nodulation, and pod formation. Expression profiles under various abiotic and biotic stress conditions reveal their involvement in stress adaptation mediated by hormone signaling pathways. Functional studies involving gene overexpression, gene knockout, and omics-based analyses highlight their contributions to cell expansion, stress tolerance, and the regulation of root system architecture. A case study focusing on soybean illustrates how differential expression and transgenic validation of expansin genes influence drought resistance and nodulation. This study lays the foundation for understanding the functions of expansin genes in legumes and provides prospects for their application in molecular breeding, genome editing, and improving stress tolerance in legume crops. Keywords Expansin proteins; Legumes; Gene expression dynamics; Evolutionary analysis; Stress response 1 Introduction In the growth regulation of plant cell walls, a type of substance called blotin has long been regarded as a key player. They do not break the components of the cell wall through hydrolysis, but rather help the cell wall extend in a specific way under acidic conditions by breaking non-covalent bonds. This type of protein is not just one or two, but A superfamily consisting of four subclasses: α -butenin (EXPA), β -butenin (EXPB), butenin-like A (EXLA), and butenin-like B (EXLB). Among them, EXPA and EXPB are the most active, especially in the aspect of loosening the cell wall. They are not only involved in seed germination or pollen tube growth, but also in leaf morphology, root bud elongation, flower and fruit development, and even the process of plant shedding. But the role of expansion protein is not limited to growth. When plants are subjected to abiotic stresses such as drought, saline-alkali conditions or abnormal temperatures, they are also "showing up". Furthermore, when symbiosis with microorganisms (such as mycorrhizae and rhizobia), these proteins also play a role in communication and adaptation (Cosgrove, 2015; Mohanty et al., 2017). Especially in leguminous plants, the expression of the expansin gene is of particular concern. They are involved in many important links related to adaptation and development, such as the adjustment of cell walls, which directly affects the structure of root systems and the formation of root nodules-and root nodules are closely related to nitrogen fixation. The expression of these genes is usually tissue-specific and can flexibly adjust according to external conditions such as drought, salt content changes, and even nutritional status. Take soybeans and alfalfa as examples. The expression of their tumescent protein genes in different tissues varies significantly, and they are also upregulated when subjected to osmotic pressure or salt stress. This indicates that they do not play a passive role in stress responses. During the symbiotic process with rhizobia, they also regulate the related mechanisms of root infection and root nodule development, and this regulation is often influenced by hormone levels and environmental signals (Giordano and Hirsch, 2004; Li et al., 2014).
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 205 This study focuses on the diversity and expression changes of the expansin gene family in leguminous plants. We will systematically sort out their classification methods, evolutionary features and structural compositions, and analyze them in combination with their expression patterns during plant growth, development and under adverse conditions. Of course, leguminous elements such as root development, nodulation, and adaptation to drought or salt stress will be the focus of our attention. With the rapid development of genomics and transcriptomics, our understanding of the regulatory mechanisms and functions of these genes is gradually deepening, which provides new directions and research foundations for the molecular improvement of leguminous crops in the future. 2 Structural Diversity of the Expansin Gene Family in Legumes 2.1 Classification of expansins: α-expansins, β-expansins, expansin-like A, and expansin-like B The blotin family in leguminous plants is not A collection of a single type. In fact, it is divided into four categories according to phylogenetic relationships: α-expansin (EXPA), β -expansin (EXPB), expansin-like A (EXLA), and expansin-like B (EXLB) (Figure 1). Among them, the α and β types have the strongest presence and the most research. Functionally, they mainly involve the relaxation of cell walls and the process of plant development. In contrast, Sample A and Sample B are much quieter. Currently, our understanding of them mainly remains at the level of gene sequences, and there is a lack of experimental evidence to prove whether they truly possess cell wall activity. Take soybeans as an example, this point is quite obvious. Researchers have identified 75 expansin genes and classified them into four subfamilies, with EXPA taking the majority, followed by EXLB, then EXPB and EXLA (Wang et al., 2024a). This distribution also indirectly confirms the focus of research and function of each sub-family. 2.2 Gene duplication, divergence, and chromosomal distribution across legume genomes Why is the expansin gene family so large in leguminous plants? One major reason is gene replication. Especially tandem replication and fragment replication, these two mechanisms provide "channels" for the increase of family members. Such replication events are particularly frequent in soybeans and their wild relatives, playing a promoting role in the expansin gene families. But these genes are not evenly distributed on the chromosomes. Some chromosomes, due to the concentrated burst of tandem replication, have formed distinct gene clusters, and this concentration also brings potential functional or regulatory connections. Meanwhile, the evolution of different genes is not static-they are influenced by natural selection. Some have experienced positive selection pressure, resulting in the gradual formation of differentiated functional characteristics in some subfamilies (Zhu et al., 2014). 2.3 Conserved motifs and structural domains: insights from sequence alignment and phylogenetics Sequence analysis of the expansin family reveals a delicate balance: on the one hand, conservation, and on the other hand, differentiation. Structurally, most inflated proteins contain two key domains: DPBB_1 (a double psi β barrel domain) and CBM63 (a carbohydrate-binding module). These structures are not randomly combined but highly conservative, especially within the same subfamily. Take soybeans as an example. EXPA members often have a set of eight conserved motifs arranged in a similar order, while other subfamilies, such as EXPB, EXLA and EXLB, although they also have their own motif combinations, the patterns are significantly different. Some motifs even only appear in specific subfamilies. This "belonging only to oneself" marker not only indicates the evolutionary differentiation among them, but may also predict the functional specialization tendency (Feng et al., 2022). In other words, although they all belong to the same superfamily, the differences between different subclasses are real, reflecting their respective "personalities" and the divergence in the process of evolution. 3 Evolutionary Insights into Expansin Genes in Legumes 3.1 Phylogenetic relationships of expansin genes among legume species and other angiosperms The story of expansins actually dates back to an even earlier stage of green algae-their rudimentary forms had already emerged. As plants gradually "come ashore" and move towards terrestrial ecosystems, these genes have not been idle either, and have begun to continuously differentiate and evolve. EXPA was the first to appear among the four subfamilies, followed by EXPB, EXLA and EXLB. However, there are also many differences among
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 206 various plants. Although in legumes and other angiosperms, most of the bulking protein genes belonging to the same subfamily can cluster into one category, showing a certain degree of conservation, the boundaries between subfamilies remain quite distinct. That is to say, they look like a family on the outside, but in fact, they have been working separately for a long time. Comparative studies have shown that the number and distribution of genes in various species are not exactly the same, but the core subfamily structure remains constant, indicating that conservation and diversity are two coexisting aspects in the process of plant evolution (Li et al., 2002; Sun et al., 2021). Figure 1 Amino acid phylogenetic tree of wild soybean expansin family members (Adopted from Feng et al., 2022) 3.2 Role of gene duplication events (segmental, tandem) in the evolution of the expansin family When it comes to why the expansin gene family has become so large, it is impossible to avoid an old acquaintance-gene replication. Fragment replication and tandem replication almost constitute the main theme of the expansion of the expansin gene. This is particularly evident in the research of soybeans: nearly 70% of the expansin genes are formed through fragment replication, and tandem replication also contributes nearly 15%. Although they differ in proportion, both play a key role in "creating" diversity. Interestingly, these replications do
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 207 not occur alone in one place. They often concentrate in specific regions on chromosomes and gradually form gene clusters. This local "clustering" not only enables these genes to be preserved but also provides opportunities for subsequent functional differentiation. Thus, the abilities to adapt to developmental needs and cope with environmental changes were gradually established (Li et al., 2024; Wang et al., 2024c). 3.3 Selective pressures and evolutionary rates inferred from Ka/Ks analyses Not all genes function freely during the process of evolution. In fact, most of the genes for bloating proteins are very "regular". From the analysis of the Ka/Ks ratio, it can be seen that for both soybeans and two-grain wheat, the majority of their tumescent protein gene pairs show a Ka/Ks ratio less than 1, indicating that they have undergone purification selection and tend to retain their original functions with few modifications. But this doesn't mean there are no changes at all. Positive selection signals may appear at certain specific loci, especially in regions related to functional differences. That is to say, these places seem more willing to "take risks" and try some new changes, especially within certain specific subfamilies. The existence of such positive selection actually indirectly indicates that the expansion protein genes are not static; they are still seeking opportunities to evolve new functional possibilities (Li et al., 2023). 4 Expression Dynamics during Plant Development 4.1 Tissue-specific expression patterns in roots, stems, leaves, flowers, and nodules Different tissues "speak" in different ways during development-each tissue has its own "language", that is, a specific combination of gene expressions. In parts such as roots, stems, leaves and flowers, different genes each take the stage and perform their own functions. In model plants like Arabidopsis thaliana, through transcriptome and proteome studies, it has been found that each tissue has its own unique expression profile, which varies greatly from one another. Take Gen for example, studies on single-cell transcriptomes have revealed significant differences in gene expression among different cell types, and these differences are constantly adjusted as the development stage progresses. But not all parts are as clear as the roots. For instance, in above-ground organs such as leaves and flowers, the changes in expression are more closely related to tissue maturity. The later it goes, the more regular the expression becomes (Jean-Baptiste et al., 2019). There are also tissues such as flowers and seeds, which belong to reproductive structures. Their expression changes are often related to organ identity confirmation and maturation rhythm (Wellmer et al., 2006). 4.2 Temporal regulation during key developmental stages Gene expression is not always so "free and unrestrained". At some critical junctures, such as when seeds just germinate, organs first form, or when entering the reproductive stage, the rhythm of expression becomes particularly tight and layered. During the early embryo and germination stages, many genes suddenly "come online", with changes in expression regions and times. This transcriptional recombination is actually setting the tone for cell fate and paving the way for subsequent differentiation (Palovaara et al., 2017). However, once it comes to the development period of the floral organs, the situation is quite different. Some specific gene families are only expressed when flower buds are formed or organs are initially established, and then they fall silent. And groups of co-expressed genes act like a baton, dominating the entire process from organ initiation to maturity (Ryan et al., 2015). The development process of the stem is similar, but with a slightly different rhythm. It usually goes through a series of stages from cell division to expansion and then to secondary growth, and the transcriptome expression also changes accordingly (Zhang et al., 2024). 4.3 Correlation of expansin expression with cell expansion and morphogenesis Regarding the expansion proteins in leguminous plants, we don't have much direct data at hand yet, but some studies on model plants have already revealed quite a few clues. Cell expansion is not something that can be accomplished by a single gene working alone; it requires the collaborative efforts of an entire "team": those that regulate cell wall relaxation, maintain turgor pressure, and respond to hormone signals will all be collectively upregulated at critical stages. For instance, during the rapid growth period of leaves, the expression levels of those genes related to the cell wall are often very high, especially those responsible for wall structure adjustment
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 208 (Mergner et al., 2020). This type of expression dynamics is highly consistent with the stage of rapid cell volume growth and organ formation, and also indirectly indicates that the participation of expansion proteins in the development process is actually quite high-especially in morphological formation and structural shaping. 5 Expansin Gene Regulation under Abiotic and Biotic Stress 5.1 Expression modulation in response to drought, salinity, cold, and heat stress In the face of various abiotic stresses, plants do not just sit and wait to die. Expansin genes show significant expression changes under such environmental stress, but these changes often vary depending on the species, tissue and type of stress. In wild soybeans, the GsEXLB14 gene is activated under stress. Overexpression can promote root growth and enhance the plant's stress resistance at the same time (Figure 2). Not only soybeans, but similar examples can also be found in other plants. Once the expression of NtEXPA4 in tobacco and BrEXLB1 in Brassica plants is upregulated, proline accumulation increases and the root system becomes longer, resulting in enhanced drought and salt tolerance (Muthusamy et al., 2020). However, it is not always so direct in all cases. For instance, under cold and hot stress, the expression of blotin will also increase, but the underlying mechanism of action may be more complex. Like TaEXPB7-B in wheat, it is activated under the combined influence of low temperature and ABA (abscisic acid), which helps to enhance cold resistance and maintain growth (Feng et al., 2019). From these circumstances, it can be seen that the expression regulation of the expansion protein gene is intricately related to osmosis regulation, cell wall structure stability, and even antioxidant mechanisms (Kuluev et al., 2016; Chen et al., 2019). 5.2 Induction by pathogen infection and involvement in defense signaling As soon as a plant encounters a pathogen, its defense system begins to be activated, and the expansin gene is often involved. But things are not that simple. Sometimes their roles are even a bit "contradictory". In wild peanuts, a gene called AdEXLB8, when introduced into tobacco, can significantly enhance resistance to pathogenic bacteria and nematodes, and also improve drought tolerance. This is mainly achieved by activating jasmonic acid and ABA signaling pathways, thereby enhancing antioxidant defense capabilities (Brasileiro et al., 2021). However, this positive effect is not static. For instance, in tobacco, although NtEXPA4 can make plants more drought-resistant and salt-tolerant, it appears more "vulnerable" when facing powdery mildew or bacterial infection-infection becomes easier (AbuQamar et al., 2013; Chen et al., 2018). This indicates that there may be some kind of "tug-of-war" between immunity and growth for expansins, and it is not always the best of both worlds. 5.3 Crosstalk with plant hormones (ABA, auxin, ethylene) in stress adaptability When plants are under stress, hormone signaling pathways are almost impossible to be absent. The expression changes of the expansin gene are largely regulated by abscisic acid, auxin and ethylene. These three hormones play different roles in stress responses, but all can influence the behavior of dilator proteins. Abolic acid, which has long been regarded as the "big boss" of drought and salt resistance responses, can induce the expression of genes such as TaEXPA2 (wheat) and OfEXLA1 (osmanthus), thereby helping plants enhance their adaptability (Dong et al., 2023). Auxin related dilating proteins, on the other hand, are more inclined to promote root growth and cell expansion, representing a more "growth regulation" pathway. The role of ethylene is slightly complex. It is involved in regulating cell wall remodeling. For example, the upregulation of GsEXLB14 when there is insufficient water can help maintain the extended state of cells (Han et al., 2012). The three do not operate independently but interweave and collaborate to jointly determine whether the plant maintains its state, accelerates growth, or adjusts its shape under stress. This coordination ability is essentially a manifestation of plant adaptability and also indicates that the regulation of expansion proteins is far more complex than it appears on the surface. 6 Functional Characterization of Expansin Genes 6.1 Gene knockout and overexpression studies in model and crop legumes What exactly can expansive protein do? The most direct way is to "turn them on" or "turn them off" to see the effect. Through the research on gene knockout and overexpression, many problems have begun to become clear
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 209 (Chen, 2024; Wang et al., 2024b). For instance, in wild soybeans, the overexpression of GsEXPB1 has led to an increase in the number and length of roots, as well as a significant rise in weight. Not only that, but its tolerance to salt stress has also increased. This change is quite revealing-the connection between the activity of expansins and the improvement of root structure and stress adaptation is indeed not accidental. Similar effects have also been observed in other plants. For instance, in tobacco and Brassica plants, once some blotting protein genes (such as NtEXPA4, NtEXPA11, BrEXLB1) are highly expressed, they can push the root system to break through the soil downward, and also help maintain ion balance, enhancing the plant's drought and salt resistance. However, if we knock out certain genes, such as OsEXPA10 in rice, the result changes-the elongation of root cells is significantly reduced, indicating that these genes are actually "indispensable" for normal development (Che et al., 2016). Figure 2 Phenotype of soybean hairy roots overexpressing GsEXLB14 under normal, salt, and drought stress conditions (Bar = 2 cm) (Adopted from Wang et al., 2024a) 6.2 Functional assays linking expansins to cell wall loosening and root architecture What exactly is expansin doing? They do not directly determine how cells grow, but they play a significant role in the "loosening" of cell walls, which is precisely the key link for cells to expand and organs to elongate. Some short-term expression experiments and pilous root transformation studies have found that the expansion protein is indeed located within the cell wall. For instance, when GsEXPB1 of soybeans is overexpressed in hairy roots, the roots not only grow faster but also can better cope with salt stress. Not only that, experiments on other model plants such as Arabidopsis thaliana have also made similar findings-seeds germinate more easily, root hairs grow
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 210 more, and even the overall growth of roots has improved. Behind these phenomena, almost all of them are inseparable from the "behind-the-scenes actions" of bloating proteins in cell wall modification (Dabravolski and Isayenkov, 2025). Judging from these results, whether in normal development or under adverse conditions, blotin seems to be helping cells "unbind", allowing plants to cope with the environment more calmly. 6.3 Omics approaches (transcriptomics, proteomics) identifying candidate expansins in stress and development Not every expansion protein can be immediately identified for its function, especially during complex stress responses or developmental processes. At this point, omics technology comes into play. Like in wild soybeans, transcriptome analysis revealed that some expansins were specifically induced to be expressed under conditions such as salt, drought, and cold, and this expression often carried organ specificity. Promoter analysis further tells us that their regulation is not only dependent on environmental stimuli, but also involves developmental processes, hormone signals, and even adverse cis-regulatory elements (Chen et al., 2020). Proteomics and co-expression networks have not been idle either. Research results indicate that a batch of candidate genes for bulking proteins may be involved in processes such as cell wall remodeling, antioxidant defense, and osmotic regulation. Although these data cannot be used to draw a direct conclusion, they at least provide a very clear clue-we can understand the position of these genes in plant adaptability from a broader perspective, especially in leguminous crops that are particularly sensitive to environmental responses. 7 Case Study: Expansin Gene Functions in Soybean (Glycine max) 7.1 Genome-wide identification and classification of expansin genes in soybean If asked which crop has the "most complete" family of expansive proteins, soybeans must be on the list. So far, 75 expin genes have been identified and are distributed in four subfamilies: EXPA, EXPB, EXLA and EXLB. EXPA has the largest number of members, accounting for approximately two-thirds. In contrast, EXLA is relatively "less popular" and has the fewest quantity. These genes are not only numerous but also structurally interesting. Genes within the same subfamily are often highly similar in intron and exon structures and share some conserved motifs, which indicates that they are very likely to have evolved from a common "ancestral module". Of course, the formation of such diversity does not come out of thin air-fragment repetition and serial replication events are all driving the expansion and functional differentiation of this family. 7.2 Expression profiling under drought and nodulation stages reveals functional clusters The presence of expansive proteins in soybeans is not even, especially in areas such as roots and root nodules, where they are often much more active. The analysis of the expression profile reveals some patterns, but also brings out a lot of complexity. Genes like GsEXPB1 and GsEXLB14 are "resident" genes in the roots, and they will be significantly upregulated especially when encountering drought or salt stress. GmEXPB2 and GmINS1, on the other hand, are more likely to be expressed when root nodules form or when plants are deficient in phosphorus. Their changes are even associated with the size of root nodules and the enhancement of nitrogen fixation capacity. Not all expansins are activated under adverse conditions. Some only work at specific times and in specific areas. For instance, during the tumor formation stage, some genes are activated particularly early, while others "make a grand entrance". Transcriptome data further support this point-their expression is influenced by both abiotic stress and developmental stage (Li et al., 2015; Yang et al., 2021). 7.3 Transgenic approaches demonstrate roles in root elongation and abiotic stress tolerance The most direct way to figure out exactly what a certain expansin is "responsible for" is through transgenic experiments. Many studies nowadays are conducted in this way. Both transgenic experiments on the hairs of soybean roots and the entire plant have found that after overexpressing genes such as GsEXPB1, GsEXLB14, GmEXPB2 or GmEXPA7, the number, length and biomass of roots all increased, and the plant's tolerance to stress such as salt, drought and low phosphorus also improved accordingly. However, this matter is not one-sided. If we change our approach and deal with it by interfering with or inhibiting these genes, the results will soon become apparent: the roots won't grow and there will be problems with the formation of nodules. For instance, GsEXPB1
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 211 not only promotes root growth but also enhances root tolerance under salt stress (Figure 3). The effect of GmEXPB2 is more comprehensive. It not only promotes root elongation but also improves root nodule formation and phosphorus absorption capacity (Zhou et al., 2014; Kong et al., 2019). In other words, these genes are not merely "auxiliary tools", but a core link in root development and stress response. Figure 3 Overexpression of GsEXPB1 significantly promoted cultivated soybean hairy roots growth and salt stress tolerance. (A) Construction of expression vector, P35S indicates CaMV35S promoter, HA indicates HA protein tag, NOS indicates terminator. (B) Detection results of gene transcription and protein expression in transgenic hairy roots. (C) Phenotypic observation of hairy roots overexpressing GsEXPB1 under normal growth conditions and salt stress, bar = 2 cm (Adopted from Feng et al., 2022) 8 Concluding Remarks The expansin gene in leguminous plants is complex, but there are indeed patterns to follow. They are not a type of "uniform specification" genes, but a large family with diverse structures and functions, classified into four subgroups: EXPA, EXPB, EXLA and EXLB. Many members have similar conservative domains and repetitive motifs, which look like traces left by their "ancestors". The reason why they are increasing in number is mainly driven by fragment replication and serial replication, while the subsequent functional differentiation is gradually shaped by various selective pressures.
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 212 But the "voices" of these genes are not always synchronized. At different tissues and developmental stages, their expressions have their own rhythms, especially in the root system, root nodules, and when facing adverse conditions such as drought or salinization, they are particularly active. Genes like GmEXPB2 have been repeatedly demonstrated in research that after overexpression, not only do roots grow faster and root nodules increase, but the stress resistance also improves significantly (and all of these are ultimately linked to yield). Of course, including these genes in the breeding program is not just empty talk. Whether using traditional marker-assisted selection or with the help of CRISPR/Cas, the "precise scissors", the application of expansion proteins is becoming more realistic. They may alter the configuration of roots, enhance nutrient absorption capacity, and even offer a feasible path in terms of reducing medication and increasing yield. However, behind these seemingly "promising" directions, there are still many problems to be solved. For instance, how do these genes interact with each other? Do they have a deeper integration with hormone signals or stress pathways? Which ones are the core functions and which ones are merely "background signals"? In addition, many functional verifications are still at the laboratory stage, and the stability of the field environment and the applicability across varieties have not been fully tested. Future research may need to delve more into these cross-regions. It is not only about functional verification, but also delves into multiple levels such as transcription, protein, and metabolism. It is not only necessary to look at short-term phenotypes, but also to observe long-term adaptability. Only when we truly put these laboratory achievements into the fields for verification can they possibly become truly useful breeding resources, rather than just highlights in papers. Acknowledgments I am very grateful to Ms. Fan for her meticulous review of the manuscript and her suggestions for improvement of the logical coherence. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References AbuQamar S., Ajeb S., Sham A., Enan M., and Iratni R., 2013, A mutation in the expansin-like A2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana, Molecular Plant Pathology, 14(8): 813-827. https://doi.org/10.1111/mpp.12049 Brasileiro A., Lacorte C., Pereira B., Oliveira T., Da Silva Ferreira D., Mota A., Saraiva M., Araújo A., Silva L., and Guimaraes P., 2021, Ectopic expression of an expansin-like B gene from wild Arachis enhances tolerance to both abiotic and biotic stresses, The Plant Journal, 107(6): 1681-1696. https://doi.org/10.1111/tpj.15409 Che J., Yamaji N., Shen R., and Ma J., 2016, An Al-inducible expansin gene, OsEXPA10 is involved in root cell elongation of rice, The Plant Journal, 88(1): 132-142. https://doi.org/10.1111/tpj.13237 Chen Q.S., 2024, Genome-wide association studies in fabaceae: progress and prospects, Genomics and Applied Biology, 15(4): 212-222. https://doi.org/10.5376/gab.2024.15.0023 Chen L., Zou W., Fei C., Wu G., Li X., Lin H., and Xi D., 2018, α-Expansin EXPA4 positively regulates abiotic stress tolerance but negatively regulates pathogen resistance in Nicotiana tabacum, Plant and Cell Physiology, 59(11): 2317-2330. https://doi.org/10.1093/pcp/pcy155 Chen S., Luo Y., Wang G., Feng C., and Li H., 2020, Genome-wide identification of expansin genes in Brachypodium distachyon and functional characterization of BdEXPA27, Plant Science, 296: 110490. https://doi.org/10.1016/j.plantsci.2020.110490 Chen Y., Zhang B., Li C., Lei C., Kong C., Yang Y., and Gong M., 2019, A comprehensive expression analysis of the expansin gene family in potato (Solanum tuberosum) discloses stress-responsive expansin-like B genes for drought and heat tolerances, PLoS ONE, 14(7): e0219837. https://doi.org/10.1371/journal.pone.0219837 Cosgrove D., 2015, Plant expansins: diversity and interactions with plant cell walls, Current Opinion in Plant Biology, 25: 162-172. https://doi.org/10.1016/j.pbi.2015.05.014 Dabravolski S., and Isayenkov S., 2025, Expansins in salt and drought stress adaptation: from genome-wide identification to functional characterisation in crops, Plants, 14(9): 1327. https://doi.org/10.3390/plants14091327
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 213 Dong B., Wang Q., Zhou D., Wang Y., Miao Y., Zhong S., Fang Q., Yang L., Xiao Z., and Zhao H., 2023, Abiotic stress treatment reveals expansin like A gene OfEXLA1 improving salt and drought tolerance of Osmanthus fragrans by responding to abscisic acid, Horticultural Plant Journal, 10(2): 573-585. https://doi.org/10.1016/j.hpj.2022.11.007 Feng X., Li C., He F., Xu Y., Li L., Wang X., Chen Q., and Li F., 2022, Genome-wide identification of expansin genes in wild soybean (Glycine soja) and functional characterization of Expansin B1 (GsEXPB1) in soybean hair root, International Journal of Molecular Sciences, 23(10): 5407. https://doi.org/10.3390/ijms23105407 Feng X., Xu Y., Peng L., Yu X., Zhao Q., Feng S., Zhao Z., Li F., and Hu B., 2019, TaEXPB7-B, a β-expansin gene involved in low-temperature stress and abscisic acid responses, promotes growth and cold resistance in Arabidopsis thaliana, Journal of Plant physiology, 240: 153004. https://doi.org/10.1016/j.jplph.2019.153004 Giordano W., and Hirsch A., 2004, The expression of MaEXP1, a Melilotus alba expansin gene, is upregulated during the sweetclover-Sinorhizobium meliloti interaction, Molecular Plant-Microbe Interactions, 17(6): 613-622. https://doi.org/10.1094/MPMI.2004.17.6.613 Han Y., Li A., Li F., Zhao M., and Wang W., 2012, Characterization of a wheat (Triticum aestivum L.) expansin gene, TaEXPB23, involved in the abiotic stress response and phytohormone regulation, Plant Physiology and Biochemistry, 54: 49-58. https://doi.org/10.1016/j.plaphy.2012.02.007 Huang L., and Schiefelbein J., 2015, Conserved gene expression programs in developing roots from diverse plants, Plant Cell, 27: 2119-2132. https://doi.org/10.1105/tpc.15.00328 Jean-Baptiste K., McFaline-Figueroa J., Alexandre C., Dorrity M., Saunders L., Bubb K., Trapnell C., Fields S., Queitsch C., and Cuperus J., 2019, Dynamics of gene expression in single root cells of Arabidopsis thaliana, The Plant Cell, 31: 993-1011. https://doi.org/10.1105/tpc.18.00785 Kong Y., Wang B., Du H., Li W., Li X., and Zhang C., 2019, GmEXLB1, a soybean expansin-like B gene, alters root architecture to improve phosphorus acquisition in Arabidopsis, Frontiers in Plant Science, 10: 808. https://doi.org/10.3389/fpls.2019.00808 Kuluev B., Avalbaev A., Mikhaylova E., Nikonorov Y., Berezhneva Z., and Chemeris A., 2016, Expression profiles and hormonal regulation of tobacco expansin genes and their involvement in abiotic stress response, Journal of Plant Physiology, 206: 1-12. https://doi.org/10.1016/j.jplph.2016.09.001 Li M., Liu T., Cao R., Cao Q., Tong W., and Weining S., 2023, Evolution and expression of the expansin genes in emmer wheat, International Journal of Molecular Sciences, 24(18): 14120. https://doi.org/10.3390/ijms241814120 Li X., Tan Z., Zeng R., and Liao H., 2015, GmEXPB2, a cell wall β-expansin, affects soybean nodulation through modifying root architecture and promoting nodule formation and development, Plant Physiology, 169: 2640-2653. https://doi.org/10.1104/pp.15.01029 Li X., Walk T., and Liao H., 2014, Characterization of soybean β-expansin genes and their expression responses to symbiosis, nutrient deficiency, and hormone treatment, Applied Microbiology and Biotechnology, 98: 2805-2817. https://doi.org/10.1007/s00253-013-5240-z Li Y., Darley C., Ongaro V., Fleming A., Schipper O., Baldauf S., and McQueen-Mason S., 2002, Plant expansins are a complex multigene family with an ancient evolutionary origin, Plant Physiology, 128: 854-864. https://doi.org/10.1104/pp.010658 Li Y., Zhang Y., Cui J., Wang X., Li M., Zhang L., and Kang J., 2024, Genome-wide identification, phylogenetic and expression analysis of expansin gene family in Medicago sativa L., International Journal of Molecular Sciences, 25(9): 4700. https://doi.org/10.3390/ijms25094700 Mergner J., Frejno M., Messerer M., Lang D., Samaras P., Wilhelm M., Mayer K., Schwechheimer C., and Kuster B., 2020, Proteomic and transcriptomic profiling of aerial organ development in Arabidopsis, Scientific Data, 7: 334. https://doi.org/10.1038/s41597-020-00678-w Mohanty S., Arthikala M., Nanjareddy K., and Lara M., 2017, Plant-symbiont interactions: the functional role of expansins, Symbiosis, 74: 1-10. https://doi.org/10.1007/s13199-017-0501-8 Muthusamy M., Kim J., Yoon E., Kim J., and Lee S., 2020, BrEXLB1, a Brassica rapa expansin-like b1 gene is associated with root development, drought stress response, and seed germination, Genes, 11(4): 404. https://doi.org/10.3390/genes11040404 Palovaara J., Saiga S., Wendrich J., Wendrich J., Hofland N., Schayck J., Hater F., Mutte S., Sjollema J., Boekschoten M., Hooiveld G., and Weijers D., 2017, Transcriptome dynamics revealed by a gene expression atlas of the early Arabidopsis embryo, Nature Plants, 3: 894-904. https://doi.org/10.1038/s41477-017-0035-3 Ryan P., Ó’Maoiléidigh D., Drost H., Kwaśniewska K., Gabel A., Grosse I., Graciet E., Quint M., and Wellmer F., 2015, Patterns of gene expression during Arabidopsis flower development from the time of initiation to maturation, BMC Genomics, 16: 488. https://doi.org/10.1186/s12864-015-1699-6
Legume Genomics and Genetics 2025, Vol.16, No.5, 204-214 http://cropscipublisher.com/index.php/lgg 214 Sun W., Yu H., Liu M., Ma Z., and Chen H., 2021, Evolutionary research on the expansin protein family during the plant transition to land provides new insights into the development of Tartary buckwheat fruit, BMC Genomics, 22: 252. https://doi.org/10.1186/s12864-021-07562-w Wang L., Zhang T., Li C., Zhou C., Liu B., Wu Y., He F., Xu Y., Li F., and Feng X., 2024a, Overexpression of wild soybean expansin gene GsEXLB14 enhanced the tolerance of transgenic soybean hairy roots to salt and drought stresses, Plants, 13(12): 1656. https://doi.org/10.3390/plants13121656 Wang X.M., Qi Y.X., Sun G.H., Zhang S., Li W., and Wang Y.P., 2024b, Improving soybean breeding efficiency using marker-assisted selection, Molecular Plant Breeding, 15(5): 259-268. 10.5376/mpb.2024.15.0025 Wang Z., Cao J., Lin N., Li J., Wang Y., Liu W., Yao W., and Li Y., 2024c, Origin, evolution, and diversification of the expansin family in plants, International Journal of Molecular Sciences, 25(21): 11814. https://doi.org/10.3390/ijms252111814 Wellmer F., Alves-Ferreira M., Dubois A., Riechmann J., and Meyerowitz E, 2006, Genome-wide analysis of gene expression during early Arabidopsis flower development, PLoS Genetics, 2(7): e117. https://doi.org/10.1371/journal.pgen.0020117 Yang Z., Zheng J., Zhou H., Chen S., Gao Z., Yang Y., Li X., and Liao H., 2021, The soybean β-expansin gene GmINS1 contributes to nodule development in response to phosphate starvation, Physiologia Plantarum, 172(4): 2034-2047. https://doi.org/10.1111/ppl.13436 Zhang Y., Chen S., Chen S., Yue J., Liu Y., and Li Q., 2024, Transcriptome analysis reveals the developmental dynamic of stem in poplar, Industrial Crops and Products, 222(Part 1): 119317. https://doi.org/10.1016/j.indcrop.2024.119317 Zhou J., Xie J., Liao H., and Wang X., 2014, Overexpression of β-expansin gene GmEXPB2 improves phosphorus efficiency in soybean, Physiologia Plantarum, 150(2): 194-204. https://doi.org/10.1111/ppl.12077 Zhu Y., Wu N., Song W., Yin G., Qin Y., Yan Y., and Hu Y., 2014, Soybean (Glycine max) expansin gene superfamily origins: segmental and tandem duplication events followed by divergent selection among subfamilies, BMC Plant Biology, 14: 93. https://doi.org/10.1186/1471-2229-14-93
Legume Genomics and Genetics 2025, Vol.16, No.5, 215-224 http://cropscipublisher.com/index.php/lgg 215 Feature Review Open Access Gene Editing-Assisted Development of Herbicide-Resistant Lentils Hongpeng Wang, Shiying Yu Biotechnology Research Center, Cuixi Academy of Biotechnology, Zhuji, 311800, China Corresponding email: shiying.yu@cuixi.org Legume Genomics and Genetics, 2025 Vol.16, No.5 doi: 10.5376/lgg.2025.16.0022 Received: 16 Jul., 2025 Accepted: 02 Sep., 2025 Published: 22 Sep., 2025 Copyright © 2025 Wang and Yu, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang H.P., and Yu S.Y., 2025, Gene editing-assisted development of herbicide-resistant lentils, Legume Genomics and Genetics, 16(5): 215-224 (doi: 10.5376/lgg.2025.16.0022) Abstract Lentil (Lens culinaris) is a globally important legume crop, but its yield is increasingly constrained by weed competition and limited access to selective herbicides. This study explores how gene editing technologies, particularly the CRISPR/Cas system, can revolutionize the development of herbicide-resistant lentil varieties. We first discuss the potential mechanisms of herbicide resistance in plants, including target site resistance (TSR) and non-target site resistance (NTSR), and compare transgenic approaches with endogenous gene editing strategies. We then review advances in gene editing platforms, such as base editing and primer editing, and examine delivery systems for lentil. We focus specifically on recent advances in editing key genes, such as ALS (acetolactate synthase) and EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), followed by field evaluation of gene-edited lines. We also critically analyze regulatory frameworks, biosafety concerns, and public acceptance. A case study of imazethapyr-resistant lentil breeding in Canada showcases the practical applications and achievements of gene editing in lentil breeding. This study highlights the potential of combining gene editing with modern breeding tools to broaden the herbicide resistance spectrum, enhance sustainability, and ensure the sustainability of lentil production under changing agroecological conditions. Keywords Gene editing; Herbicide resistance; CRISPR/Cas; Lentil breeding; ALS gene 1 Introduction Among the many legumes, lentils (Lens culinaris Medik.) may not be the "star players", but they still firmly hold their place globally due to their rich protein content and unique position in sustainable agriculture. However, when it comes to actual cultivation, there are also quite a few problems. For instance, lentils grow slowly in the early stage, have shallow root systems and relatively weak competitiveness, which makes them almost powerless against weeds-once weeds invade, the yield loss can be very serious (Balech et al., 2023). In the past, farmers mainly relied on manual weeding or crop rotation to control weeds, but these methods were labor-intensive and time-consuming, and large-scale promotion was not realistic. So, people turned to chemical herbicides. The effect was obvious, but the plant toxicity problem that followed caused harm to the lentils themselves-in some extreme cases, the yield could even drop by half. No one is willing to lose their crops for weeding. Against this backdrop, cultivating lentil varieties that can withstand herbicides has become an unavoidable goal. This resistant variety can be used in combination with broad-spectrum herbicides, which can kill weeds without accidentally harming the lentils themselves. Naturally, it is expected to increase yields and reduce costs. However, traditional breeding methods have always been limited in this regard: there is too little natural variation, the traits are complex, and the progress is slow. Even mutant breeding or transgenic methods are often criticized-either for their low efficiency or being blocked by policies and public opinion (Singh et al., 2021). Nowadays, gene editing technology, especially CRISPR/Cas9, seems to have reached a turning point (Wang et al., 2024). It operates more precisely and efficiently, and has the potential to bypass the genetically modified label-a feature that has already shown initial success in many crops (Kuang et al., 2024). This also makes people look forward to its application on lentils. The focus of this study is precisely to sort out the latest progress of gene editing in the development of herbicide-resistant lentils. We will elaborate from several aspects: First, we will introduce the basic situation of lentil production and its vulnerability in the use of herbicides; Then explore the genetic basis of its resistance traits;
Legume Genomics and Genetics 2025, Vol.16, No.5, 215-224 http://cropscipublisher.com/index.php/lgg 216 Then focus on the practical application of technologies such as CRISPR/Cas9 in the targeted improvement of lentil traits; Finally, discuss how to incorporate these techniques into the lentil breeding system, as well as the challenges and possibilities faced. We have particularly referred to the latest achievements in lentils and related crops, attempting to present a perspective that is both realistic and forward-looking to explore how lentils can achieve more sustainable development driven by technology. 2 Herbicide Resistance Mechanisms in Plants 2.1 Target site resistance (TSR) There are many ways for plants to develop resistance to herbicides, and among them, the most frequently occurring one is the so-called target resistance (TSR). In layman's terms, it means that herbicides can no longer find a "target". This type of resistance usually occurs when there is a "mistake" in the protein-coding gene of the target of herbicide action-not a loss of function, but a change in the appearance of the key part. If a mutation occurs at a certain site in the acetyllactate synthase (ALS) gene, the result is that the herbicides that were supposed to inhibit it fail. The same situation also occurs in genes such as ACCase or EPSPS, and their mutations can also render specific herbicides ineffective (Wei et al., 2022). But interestingly, most of these mutations are not "destructive", but rather like non-synonymous SNPS or small insertions/deletions that change the lock but do not affect the normal opening of the door. Nowadays, CRISPR/Cas9 and base editing tools are transforming these mutations from "accidental occurrence" to "targeted design", significantly enhancing breeding efficiency (Tian et al., 2018). 2.2 Non-target site resistance (NTSR) Not all plant resistance to drugs depends on "modifying the target". Sometimes, plants even quietly get rid of herbicides before they reach their targets-this is non-target resistance (NTSR). This resistance does not take the "direct confrontation" route but rather relies on various detour methods: some make it difficult for the herbicide to be absorbed, some prevent it from entering the cell transport channels, and others directly isolate it within the plant (Gaines et al., 2020). More commonly, it is through metabolic means to "defuse the crisis", such as cytochrome P450, glutathione S-transferase and glycosyltransferase. These enzymes are like detoxification factories in plants, breaking down herbicides in advance. These mechanisms often involve the collaborative work of multiple genes, unlike TSR which can be resolved by a single "key mutation", making breeding more challenging. Even so, with the development of genomic tools and editing methods, achieving targeted regulation in the future may not be out of reach (Dong et al., 2021). 2.3 Transgene-based resistance vs. endogenous gene editing Using foreign genes to endow plants with herbicide resistance is no longer a novelty-in the early years, the EPSPS or bar genes of bacteria were transferred into crops in this way. This approach is direct and effective, but it cannot avoid the label of genetically modified organisms. This has kept it stuck at the threshold of regulatory approval and public opinion for a long time (Hussain et al., 2021). In contrast, endogenous gene editing sounds much more low-key. It is not "borrowing genes", but making some "fine-tuning" to the existing genes of the plant itself, such as directly modifying target genes like ALS, ACCase or EPSPS (Figure 1) (Wang et al., 2020). Technical means include CRISPR/Cas9, base editing, and even oligonucleotide-induced mutations. Because no exogenous fragments are introduced, this method faces much less regulatory pressure and is more easily accepted by the market. Some current achievements also prove that this strategy is not only applicable to model plants, but also feasible in crops such as lentils, and the effect is stable, heritable and free of genetically modified labels. 3 Gene Editing Technologies for Lentil Improvement 3.1 CRISPR/Cas-based platforms The popularity of CRISPR/Cas9 is no accident. Simple, precise and highly efficient-these features have enabled it to quickly gain a firm foothold in plant gene editing. In leguminous plants, even for crops with weak technical foundations like lentils, some people have begun to attempt to use CRISPR/Cas9 to specifically knock in or knock out target genes, especially to modify traits such as herbicide resistance (Ahmar et al., 2020). To be honest, there
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